Dysfunctional p53 formation and activity can result from aberrant expression and subcellular localization of distinct p53 isoforms or aggregates. Endometrial carcinoma (EC) is a cancer type in which ...p53 status is correlated with prognosis, and TP53 mutations are a frequent genetic modification. Here we aimed to evaluate the expression patterns of different p53 isoforms and their contributions to the formation and subcellular localization of p53 amyloid aggregates in both EC and endometrial nontumor cell lines. We found that full-length (fl) p53 and a truncated p53 isoform, Δ40p53, resulting from alternative splicing of exon 2 or alternative initiation of translation at ATG-40, are the predominantly expressed p53 variants in EC cells. However, Δ40p53 was the major p53 isoform in endometrial nontumor cells. Immunofluorescence assays revealed that Δ40p53 is mainly localized to cytoplasmic punctate structures of EC cells, resembling solid-phase structures similar to those found in neurodegenerative pathologies. Using light-scattering kinetics, CD, and transmission EM, we noted that the p53 N-terminal transactivation domain significantly reduces aggregation of the WT p53 DNA-binding domain, confirming the higher aggregation tendency of Δ40p53, which lacks this domain. This is the first report of cytoplasmic Δ40p53 in EC cells being a major component of amyloid aggregates. The differential aggregation properties of p53 isoforms in EC cells may open up new avenues in the development of therapeutic strategies that preferentially target specific p53 isoforms to prevent p53 amyloid aggregate formation.
The PI3K/Akt and Wnt/β-catenin pathways play an important role in the acquisition of the malignant phenotype in cancer. However, there are few data regarding the role of the interplay between both ...pathways in colorectal cancer (CRC) progression. The mutational status and the clinicopathological characteristics of PI3K/Akt and Wnt/β-catenin pathways were accessed by bioinformatic analysis whereas that the impact of the interplay between the activity of both pathways to explain tumorigenic potential was performed in vitro using IGF-1 and Wnt3a treatments in CRC cell models. The mutational status of these pathways did not influence the survival of CRC patients, but an association between clinicopathological characteristics in patients with mutations in one, but not in both pathways was observed. A potentiating effect on the activation of both pathways and enhanced cellular migration and proliferation was observed when both pathways were activated simultaneously with IGF-1 and Wnt3a. In addition, these effects were hindered after pretreatment with LY294002, a specific PI3K inhibitor, suggesting some dependence between these two signaling cascades. Our findings show that, regardless of mutational status, there is an interplay between the activity of PI3K/Akt and Wnt/β-catenin pathways that contributes to events related to CRC progression and that the reversal of such events using a PI3K inhibitor highlights the value of targeting these pathways for potential directed therapies in CRC patients.
Colorectal cancer (CRC) is frequently a lethal disease because of metastasis. Actin cytoskeletal rearrangement is an essential step in cell migration during activation of the epithelial-mesenchymal ...transition (EMT) program, which is associated with metastatic properties of cancer cells. Cofilin-1 protein modulates actin dynamics by promoting actin treadmilling, thereby driving membrane protrusion and cell migration and invasion. However, the role of cofilin-1 during EMT in CRC is unknown. Here, we show that cofilin-1 and p-cofilin-1 have distinct subcellular distribution in EMT cells, as determined by super-resolution microscopy images, indicating distinct roles in different areas of cells. Silenced cofilin-1 cells treated with TGF-β (siCofilin-1/TGF-β) evaded p-LIMK2-p-cofilin-1 status, leading to recovery of E-cadherin and claudin-3 at the cell-cell contact and their respective protein levels, actin reorganization, and decreased mesenchymal protein level. Furthermore, siCofilin-1/TGF-β cells exhibited decreased migration and invasion rates as well as MMP-2 and -9 activity and augmented focal adhesion size. The expression of an inactive phospho-cofilin-1 mimetic (S3E) reduced E-cadherin and claudin-3 in cell-cell contacts, reduced their protein levels, and increased vimentin protein. Based on our findings, we suggest that cofilin-1 is crucial to switching from epithelial to mesenchymal-like morphology and cell migration and invasion by regulating actin cytoskeleton organization through activation of RhoA-LIMK2-cofilin-1 signaling, impacting the cell-cell adhesion organization of colon cancer cells in EMT.
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•Cofilin-1 mediates EMT thought the actin cytoskeleton rearrangement•RhoA-LIMK2-cofilin-1 signaling regulates actin cytoskeleton dynamics.•Cofilin-1 signaling participates cell-cell adhesion organization/disassembly.•Cofilin is required for migration and invasion cell in EMT-cells.
Surprisingly, CMS1 had the highest expression of ANXA2, not CMS4 which is characterized with the classical markers of EMT. 12 Possible explanations for this association lie in the role of ANXA2 ...heterotetramer in immune response via plasmin activation 14 and in the relation between ANXA2 and STAT activation. 12,15–18 Also, the recently described association of ANXA2 with the immune microenvironment in hepatocellular carcinoma, might give us another clue to why. 19 Unlike previous works that used EGF, HGF or IGF-1 to induce EMT and evaluate ANXA2 expression, 9–11 our TGF-ß treatment led to elevated ANXA2 protein levels, elongated cellular morphology, E-cadherin internalization and vimentin upregulation. Being capable to interact directly with F-actin through its C-terminal portion, 25 ANXA2 promotes tumour progression through motility structures remodelling 26 or regulation of the endocytic trafficking. 27 In its heterotetrameric form with S100 proteins, ANXA2 exerts bundling activity over actin filaments. 28 Monomeric ANXA2 also caps and regulates the barbed ends of growing actin filaments interacting directly with globular actin units. 29 Using phospho-mutated isoforms of ANXA2, de Graauw et al. 30 unravelled a downstream mechanism of actin modulation through LIMK activation and posterior phosphorylation of cofilin-1 (CFL1). Studies have demonstrated that the RhoA-ROCK-LIMK pathway, responsible for CFL1 regulation, plays a pivotal role in the disassembly of junctional complexes by remodelling cortical actin and causing E-cadherin redistribution in the cell. 31 Even though CFL1 is found overexpressed in several tumours, its activation status will be the determinant in cytoskeleton rearrangement and tumoural progression. 32 CFL1 activity is regulated by LIMK1/2 proteins, its activity is inhibited by phosphorylation at serine 3, hindering its ability to bind and sever F-actin, and by phosphatase slingshot homolog 1 (SSH1) that dephosphorylates and activates CFL1. 33 To assess the role of p-CFL1, we evaluated EMT parameters in CFL1 S3E phospho-mimetic mutant. Dr Zheng's group from Johns Hopkins University, after establishing ANXA2 as a valuable pancreatic ductal adenocarcinoma target, developed a Listeria-based GVAX vaccine anti-ANXA2 that generates a T-cell tumour antigen-specific response and sensitizes PDAC to checkpoint inhibitor therapy. 39 There is still much to learn regarding ANXA2 and the pathways orchestrated by it; its involvement in pre-metastatic niche preparation through extracellular vesicles, role in chemo and radioresistance; and these just add to the expectations of promising new therapeutic approaches.
Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in 20% of all breast cancer subtypes, especially those that present the worst prognostic outcome through a very invasive and ...aggressive tumour. HCC-1954 (HER-2+) is a highly invasive, metastatic cell line, whereas MCF-7 is mildly aggressive and non-invasive. We investigated membrane proteins from both cell lines that could have a pivotal biological significance in metastasis. Membrane protein enrichment for HCC-1954 and MCF-7 proteomic analysis was performed. The samples were analysed and quantified by mass spectrometry. High abundance membrane proteins were confirmed by Western blot, immunofluorescence, and flow cytometry. Protein interaction prediction and correlations with the Cancer Genome Atlas (TCGA) patient data were conducted by bioinformatic analysis. In addition, β1 integrin expression was analysed by Western blot in cells upon trastuzumab treatment. The comparison between HCC-1954 and MCF-7 membrane-enriched proteins revealed that proteins involved in cytoskeleton organisation, such as HER-2, αv and β1 integrins, E-cadherin, and CD166 were more abundant in HCC-1954. β1 integrin membrane expression was higher in the HCC-1954 cell line resistant after trastuzumab treatment. TCGA data analysis showed a trend toward a positive correlation between HER-2 and β1 integrin in HER-2+ breast cancer patients. Differences in protein profile and abundance reflected distinctive capabilities for aggressiveness and invasiveness between HCC-1954 and MCF-7 cell line phenotypes. The higher membrane β1 integrin expression after trastuzumab treatment in the HCC-1954 cell line emphasised the need for investigating the contribution of β1 integrin modulation and its effect on the mechanism of trastuzumab resistance.
Açaí,
Mart., is a native plant from the Amazonian and is rich in several phytochemicals with anti-tumor activities. The aim was to analyze the effects of açaí seed oil on colorectal adenocarcinoma ...(ADC) cells. In vitro analyses were performed on CACO-2, HCT-116, and HT-29 cell lines. The strains were treated with açaí seed oil for 24, 48, and 72 h, and cell viability, death, and morphology were analyzed. Molecular docking was performed to evaluate the interaction between the major compounds in açaí seed oil and Annexin A2. The viability assay showed the cytotoxic effect of the oil in colorectal adenocarcinoma cells. Acai seed oil induced increased apoptosis in CACO-2 and HCT-116 cells and interfered with the cell cycle. Western blotting showed an increased expression of LC3-B, suggestive of autophagy, and Annexin A2, an apoptosis regulatory protein. Molecular docking confirmed the interaction of major fatty acids with Annexin A2, suggesting a role of açaí seed oil in modulating Annexin A2 expression in these cancer cell lines. Our results suggest the anti-tumor potential of açaí seed oil in colorectal adenocarcinoma cells and contribute to the development of an active drug from a known natural product.
REM sleep rebound is a common behavioural response to some stressors and represents an adaptive coping strategy. Animals submitted to multiple, intermittent, footshock stress (FS) sessions during 96h ...of REM sleep deprivation (REMSD) display increased REM sleep rebound (when compared to the only REMSD ones, without FS), which is correlated to high plasma prolactin levels. To investigate whether brain prolactin plays a role in stress-induced REM sleep rebound two experiments were carried out. In experiment 1, rats were either not sleep-deprived (NSD) or submitted to 96h of REMSD associated or not to FS and brains were evaluated for PRL immunoreactivity (PRL-ir) and determination of PRL concentrations in the lateral hypothalamus and dorsal raphe nucleus. In experiment 2, rats were implanted with cannulas in the dorsal raphe nucleus for prolactin infusion and were sleep-recorded. REMSD associated with FS increased PRL-ir and content in the lateral hypothalamus and all manipulations increased prolactin content in the dorsal raphe nucleus compared to the NSD group. Prolactin infusion in the dorsal raphe nucleus increased the time and length of REM sleep episodes 3h after the infusion until the end of the light phase of the day cycle. Based on these results we concluded that brain prolactin is a major mediator of stress-induced REMS. The effect of PRL infusion in the dorsal raphe nucleus is discussed in light of the existence of a bidirectional relationship between this hormone and serotonin as regulators of stress-induced REM sleep rebound.
•REM sleep deprivation associated with repeated footshock stress increased prolactin levels in the hypothalamus.•REM sleep deprivation associated with repeated footshock stress increased prolactin levels in the Dorsal Raphe nucleus.•Prolactin infusion in the Dorsal Raphe nucleus increased REM sleep time.
Abstract
Annexins are calcium-dependent phospholipid binding proteins that act on several cellular functions such as cytoskeleton organization, ion transport, and cell signalling. Due to its relation ...to the actin structure and cell proliferation; alterations in its expression have known implications in the progression of distinct cancer types. Among the proteins of the family, annexin A2 (AnxA2) is known for promoting cell proliferation, migration, and invasion when overexpressed. A recent study suggested AnxA2 as a diagnostic and prognostic biomarker for colorectal cancer. However, there is little evidence of its regulation and role in cell signalling pathways involved in colorectal cancer tumorigenesis. Our goal is to unravel the role of AnxA2 in the tumor progression and the signalling pathways involved. The total and phosphorylated (Y23 residue) AnxA2 levels were assessed in three human colorectal cancer cell lines (Caco-2, HT-29 e HCT-116), through imunoblotting. HT-29 cells, control or silenced (siRNA) for AnxA2, were submitted to TGF-β; treatment to induce characteristic epithelyal-mesenchymal transition (EMT) events. Proliferation, migration, and invasion were also assessed for these cells. Our results show that Caco-2 cells have the lowest phosphorylation level among the analyzed cells. In face TGF-β; treatment, the HT-29 cell line showed morphologic alterations characteristic to EMT, reduced E-cadherin protein levels, and increased vimentin expression. AnxA2 (total and phospho) expression was also enhanced. The loss of cell-cell contacts due to treatment was observed with the internalization of E-cadherin and AnxA2. The analysis of such internalization through Structured Illumination Microscopy (SIM) allowed us to observe intracellular colocalization between these two proteins. Proliferation assays reveal that TGF-β; has antiproliferative effects and in a less drastic form so does AnxA2 siRNA. AnxA2 silencing also reduced cell migration in Wound Healing assay, in a manner independent of TGF-β. The EMT induction through treatment also led to incresead invasion, which was inhibted with AnxA2 silencing or STA21 pretreatment (STAT3 selective inhibtor). Results suggest that AnxA2 has a role in cellular junction disassembly and enhanced invasion ability, characteristics of EMT process, and in cellular proliferation and migration.
Citation Format: Murilo Ramos Rocha, Annie Cristhine Moraes Sousa Squiavinato, Pedro Barcellos de Souza, Jose Andres Morgado Diaz. Annexin A2 as a regulator of colorectal tumorigenesis: Influence over epithelial-mesenchymal transition and invasive abilities abstract. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A84.
Abstract Aging is an independent prognostic factor for the diagnosis of melanoma. The extracellular matrix of the skin has well-documented changes with age, such as the loss of HAPLN1, which can lead ...to differential metastasis in aged individuals. The aged microenvironment can contribute to the phenotype switch, where aged individuals have smaller primary tumors but more metastases, i.e., going instead of growing. The extracellular matrix (ECM) has been shown to modulate the immune response either through direct binding of ECM proteins or through mechanotransduction, but it is unclear how aging affects this crosstalk. Our hypothesis is that the ECM can modulate immune response to melanoma and affect response to immune checkpoint blockade (ICB) therapy. Melanoma cells cultured on cell derived matrices (CDMs) from young and aged human fibroblasts show differential levels of expression of PD-L1, a ligand that binds to PD-1 on T cells to induce T cell exhaustion. Antibodies targeting PD-1/PD-L1 are one of the main ICB treatments available to melanoma. We are also investigating T cell motility on young and aged CDMs, as well as correlating T cell infiltration in murine tumors with ECM signatures as identified by second harmonic generation microscopy. Therefore, we have compiled evidence that aging affects the crosstalk between the extracellular matrix and the immune system, providing a new horizon for therapeutic improvement of response in older patients. Citation Format: Elizabeth I. Harper, Yash Chhabra, Alexis Carey, Murilo Ramos Rocha, Laura Hüser, Vania Wang, Agrani Dixit, Ashani Weeraratna. Crosstalk between the immune system and the extracellular matrix in the context of melanoma and aging abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4282.
Abstract There is documented sex disparity in cutaneous melanoma incidence and mortality, increasing disproportionately with age and in the male sex. However, the underlying mechanisms remain ...unclear; while biological sex differences and inherent immune response variability have been assessed in tumor cells, the role of the microenvironment surrounding the tumor, contextually in aging, has been overlooked. We find that skin fibroblasts undergo age-mediated changes in their proliferation, senescence, ROS levels and stress response that vary with sex. We find that aged male fibroblasts selectively drive an invasive and slow-cycling phenotype in melanoma cells in vitro by increasing AXL expression. This is also evident in syngeneic mouse models where metastasis is increased in aged male mice. Mechanistically, intrinsic aging in male fibroblasts coupled with elevated ROS promotes EZH2 decline thereby increasing BMP2 secretion, which in turn drives the slower-cycling, highly invasive, and therapy-resistant melanoma cell phenotype, characteristic of the aged male TME. Inhibition of BMP2 activity blocks the emergence of the invasive phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. Our data provides an integrated view of how age and sex of the host contributes to melanoma progression and therapy responses. Bridging this knowledge gap will improve patient stratification and assist in tailoring the therapy to the individual. Citation Format: Yash Chhabra, Mitchell Fane, Sneha Pramod, Laura Hueser, Daniel Zabransky, Vania Wang, Edwin Kumah, Alexis Carey, Elizabeth Harper, Murilo Ramos Rocha, Ashani Weeraratna. Sexual differences in the aged melanoma tumor microenvironment dictates metastasis and therapeutic responses abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5513.
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